Intra-islet α-cell Gs signaling promotes glucagon release

Glucagon, a hormone released from pancreatic α-cells, is critical for maintaining euglycemia and plays a key role in the pathophysiology of diabetes. To stimulate the development of new classes of therapeutic agents targeting glucagon release, key α-cell signaling pathways that regulate glucagon secretion need to be identified. Here, we focused on the potential importance of α-cell Gs signaling on modulating α-cell function. Studies with α-cell-specific mouse models showed that activation of α-cell Gs signaling causes a marked increase in glucagon secretion. We also found that intra-islet adenosine plays an unexpected autocrine/paracrine role in promoting glucagon release via activation of α−cell Gs-coupled A2A adenosine receptors. Studies with α-cell-specific Gαs knockout mice showed that α-cell Gs also plays an essential role in stimulating the activity of the Gcg gene, thus ensuring proper islet glucagon content. Our data suggest that α-cell enriched Gs-coupled receptors represent potential targets for modulating α-cell function for therapeutic purposes.

Major comments: 1.The Gcg Cre utilized here has not been validated for brain expression.Gcg neurons are known to be expressed in the hindbrain and function to regulate various physiological functions including feeding patterns, body weight in some cases, motivation for feeding etc.More and more studies are being published on the impact of these neurons on physiology.Therefore, validation that the genetic manipulation has not targeted the brain is required.They easily could have done the HA tag staining should have been completed in the nucleus of the solitary tract-why it was done in the hypothalamus instead is unclear.2. Were the data in Figure 1 analyzed with an ANOVA for repeated measures?If there is a group effect, but no time effect then statistically you can't say that DCZ actually increased glucagon or insulin.The statistics need to be redone with an ANOVA for repeated measures with an appropriate post hoc analysis for all graphs where time is a variable.3. Figure 1: Although it is not clear whether this is statistically significant, there is a 50% drop in glucagon with saline injection and with DCZ in the control animals.This drop is prevented in the GsD animals with DCZ-again it is not clear whether DCZ actually significantly increases glucagon at 15 min or whether it is simply preventing a drop in glucagon.This finding is very curious and needs further exploration/explanation. 4. Figure 1: Is there a statistical increase in glucose with the saline or DCZ injection?If so, then these studies are likely confounded by the animal's stress levels.5. Figure 1 and Supp Figure 1, perifusion studies: Is the relative increase in glucagon with the Ala or AAM greater than the control animals?It seems not.Thus, the increase in glucagon is simply due to an increase in basal concentrations.If this is the case then there seems to be an ex vivo and in vivo difference in glucagon responses that needs to be discussed.Further, it is unclear why a doubling of glucagon under "basal conditions" is visible in perifused islets but not in vivo.The fact that the insulin is normal at baseline and is higher with amino acid stimulus even though the relative increase in glucagon is similar dissociates glucagon and insulin responses to amino acids under these conditions.This becomes hard to see with the expanded scale in Figure 1g.The components of the perifusion experiments should be separated out so the data can be visualized on an appropriate scale for Fig 1g .In fact, is it the scale or did the control alpha cells not increase glucagon secretion in response to alanine? 6. Statistical analysis of the perifusion studies needs to be reported.7. Body weight of Adora2af/f Gcg cre mice is needed.8. Interpretation of the 2dg data in the alpha-A2AR KO mice is not accurate.These mice had decreased glucagon and increased glucose responses to 2DG.Under glucoprivic conditions, increases in glucagon function to stimulate hepatic glucose production and increase plasma glucose.Since insulin levels are the same, then this clearly indicates that the insulin to glucagon ratio should favor a suppression of hepatic glucose production.Therefore, some other counterregulatory factor is regulating the glucose response to 2DG in the face of inadequate glucagon levels in these animals.This suggests off-target impact on sympathetic neurons.Plasma catecholamines, which also potently respond to 2DG and regulate glucose homeostasis should be assessed.9.There are more 2DG interpretive issues in Figure 7. Specifically in the second part of this sentence: "The glucopenia-induced increases in blood glucose levels were significantly more pronounced in a-GsKO mice (Fig. 7d), probably due to decreased plasma insulin levels (Supplementary Fig. 7a, b)."The 2dg-induced (and it's not 2DG it's the increase in glucose that drives the increase in insulin) insulin secretion was not reduced, it simply returned to baseline quicker.Thus, the conclusion thatα-cell Gs signaling contributes to the counterregulatory glucagon responses is not accurate.This is another specific example where a repeated measures anova for time is necessary so that the responses over time can be accurately assessed and interpreted.10.The interpretation of the glucagon responses in the alpha-GsKO mice is also curious.Gcg expression is decreased, islet glucagon content is decreased.So if there is limited gene and protein, then it is not surprising that there is no glucagon response to glucagon secretagogues.It also brings to question why the KO of alpha cell Gs leads to a decrease in Gcg expression in the first place.

General response by the authors
We would like to thank the three reviewers for their constructive comments and suggestions.We appreciate their time and effort and believe that this manuscript has been significantly improved based upon their suggestions.
Please note that we carried out many additional experiments to address the various comments raised by the reviewers.The additional work that we performed has led to the incorporation of several new figure panels (including Suppl.Figures) into the revised version of the manuscript.
Our response to the comments of the three reviewers (highlighted in yellow)

Reviewer #1
In this study, Liu et al have generated a number of new mouse strains to clarify the role of the Gs protein and Gs-coupled receptors in the regulation of glucose homeostasis and glucagon secretion.Chemigenetic Gs-coupled DREADD receptors were specifically expressed in alpha cells and their activation improved glucose tolerance and promoted both glucagon and insulin secretion in vivo and enhanced hormone release from islets ex vivo in response to alanine and depolarization.Pharmacological and genetic tools were used to demonstrate a role for adenosine A2A receptors to sustain glucagon secretion under hypoglycemic conditions, an effect proposed to depend on auto-or paracrine effects from adenosine released within islets.Consistent with the importance of Gs-coupled receptor signaling, lack of the Gs protein in alpha cells was associated with impaired glucagon secretion as well as reduced glucagon gene transcription.This is a sound and interesting study providing valuable new insights into the regulation of glucagon secretion.An extensive amount of data is presented to support the conclusions.The methodology is solid, the experiments are overall well-designed and the results clearly described.There are nevertheless some points that should be considered for improving the manuscript.
Thank you for your very positive comments about our manuscript.
1. P 5: The reader may get the impression that the authors discovered the selective expression of A2AR in alpha cells, but this information was actually picked up from already published data.Moreover, as can be seen in Fig 4a and Suppl Fig 3a, the Adora2 receptor is expressed also in pancreatic delta cells (to almost the same extent as in alpha cells), which may be worth mentioning.
As requested by the reviewer, we clarify in the revised manuscript that the expression of the A2AR in -cells was 'picked up' from published data and that this receptor subtype is also expressed in δ-cells.Specifically, we made the following changes: Results section (page 11, 2nd paragraph): "Analysis of previously published scRNAseq data from human and mouse islets 1,2 led to the identification of three Gs-coupled receptors that are selectively expressed in α-cells, as compared to other islet cell types."In the revised manuscript, we now also mention that the A2AR is also expressed in -cells (page 11, center): "This receptor subtype is also expressed at low to moderate levels in mouse islet δcells (Supplementary Fig. 3a As requested by the reviewer, we now provide a significantly improved version of Supplementary Fig. 1b.For improved clarity, we now show the various channels used.

P 8:
The authors claim that DCZ enhanced glucagon secretion at both 3 and 12 mM glucose but this is not evident from Fig 1g .The difference exists already before application of DCZ and the effect of acute application of the drug is not at all evident and no statistics is presented to support the statement.
We subjected the data shown in Fig. 1g and all other islet perifusion data to a thorough statistical analysis.To correct for differences in basal hormone secretion, we calculated area of the curve (AOC) values by subtracting the areas under or over the baseline 3 .This type of analysis is the method of choice when baseline levels between two or more experimental groups differ 3 .We found that DCZ treatment of -GsD islets resulted in a significant increase in glucagon secretion only in the presence of alanine (Fig. 1g, h).Under physiological conditions, pancreatic islets are exposed to high levels of circulating amino acids which increase the responsiveness of -cells to various glucagon secretagogues 4,5 .This observation may explain why DCZ treatment of -GsD islets had no significant effect on glucagon release in the absence of alanine (new Fig. 1g, h).
In the new version of the manuscript, we added AOC bars next to Fig. 1g (new Fig. 1h) and subjected these data to a statistical analysis.Moreover, we subjected all other islet perifusion data to the same type of analysis (please see the revised versions of Fig. 1g-j; Fig. 3a, b, e, h-j; Fig. 4b-d; Fig. 5d-f; Fig. 7h, and Suppl.Fig. 1 k, l).We added the following additional text to the Results section (page 9): "DCZ (10 nM) treatment of -GsD islets in the presence of 3 or 12 mM glucose resulted in significant increases in glucagon secretion only in the presence of alanine (3 mM) (Fig. 1g, h).Under physiological conditions, pancreatic islets are exposed to high levels of circulating amino acids which increase the responsiveness of -cells to various glucagon secretagogues 4,5 , providing a likely explanation for the inability of DCZ to stimulate glucagon release from -GsD islets in the absence of alanine (Fig. 1g, h)." 4. P 8, 3rd line from the bottom: It is mentioned that GsD shows enhanced basal activity in vitro, but Fig 1d indicates that there is also higher basal glucagon levels in vivo.
Thank you for raising this point.Although there was a trend towards higher basal plasma glucagon level in -GsD mice (time 0), this effect failed to reach statistical significance (p= 0.085; (p= 0.085; Student's t-test at time 0).We now mention this fact in the revised version of the manuscript (page 7, bottom). 5. P 11, 2nd para: Treatment of A2AR agonist is said to have little effect on cAMP at low glucose but from Fig 3c it seems that there is reduction, similar in magnitude to the increase observed at 12 mM glucose.
We fully agree with the reviewer.We should have addressed this issue in more detail in the original manuscript.We now clarify this matter in the revised version of the manuscript (page 12, 2nd paragraph): "We found that A2AR agonist treatment (UK 432097, 5 or 20 nM) of α-CAMPER islets resulted in a small reduction in cAMP levels at G3 but caused a significant increase in cAMP accumulation at G12 (Fig. 3c, d).The A2AR agonist-induced decrease in cAMP levels at G3 is probably due to the facts that A2ARs are already strongly stimulated by high endogenous adenosine levels 6 and that -cell cAMP levels are already high in a low glucose environment 7 .In agreement with these observations, addition of the A2AR antagonist SCH 442416 (0.5 mM) led to a very robust reduction of cAMP levels at G3 (Fig. 3c), raising the possibility that the inhibitory effect of the A2AR agonist at G3 was caused by A2AR desensitization or the activation of other, yet unknown, signaling pathways that interfere with adenosine-induced cAMP production."The detailed cellular mechanisms underlying this phenomenon remain to be explored experimentally in future studies 6. P 11, 2nd para: The authors speculate that cAMP levels in alpha cells are already high at low compared to high glucose.Studies of the effects of glucose on alpha cell cAMP has shown that it is indeed the case, and the authors may consider to include a reference, such as PMID 30953108.
Thank you for directing us to this highly relevant paper (new ref. 41).We now cite this work in the revised version of the manuscript (page 12, center, and Discussion section, page 24, bottom).3c and 4b and 4c, no or only one data point is shown before the application of the test stimulus, which makes it impossible to evaluate the effects.

In Figs
Very good point.As requested by the reviewer, we measured glucagon release before the application of the test stimulus using samples stored in the freezer.We modified Fig. 3 c and 4b,  c, as requested by the reviewer.

P 15/Fig 4g,h: 2-DG administration triggers glucagon secretion
, which is impaired in the a-A2AR-KO.Yet, the resulting hyperglycemia is more pronounced in the KO, despite similar insulin levels.This seemingly confusing observation deserves to be discussed.
Like the reviewer, we were also puzzled by these findings.For this reason, we decided to repeat this experiment with a new set of mice that we specifically generated to revisit this issue.Using this new batch of mice, we were unable to replicate the data presented in original version of the manuscript (Fig. 4g, h; probably an artifact).The new data show that 2-DG-induced increases in plasma glucagon, plasma insulin, and blood glucose levels were not significantly affected by the lack of -cell A2ARs.We therefore replaced Fig. 4g, h (original manuscript) with a new figure displaying these new data (Suppl.Fig. 4q-s) in the revised manuscript).We also changed the text in the Results section accordingly in the revised manuscript (page 16, center): " We found that 2-DG-induced increases in plasma glucagon, plasma insulin, and blood glucose levels were not significantly affected by the lack of -cell A2ARs (Supplementary Fig. 4q-s)."9. Investigating the role of Gs in the a-GsKO, the authors focused on beta adrenergic signaling using isoproterenol.That is interesting but it would have been logical to also test an A2AR agonist to consolidate the findings from the A2AR-KO islets.
To address this point, we treated α-GsKO and control islets with a selective A2AR agonist (UK 432097, 50 nM; revised Fig. 3f, g).We found that UK 432097-induced glucagon secretion was absent or nearly completely abolished in α-GsKO islets at both G3 and G12, respectively.In addition, we carried out vivo experiments with PSB 0777 (1 mg/kg, i.p.), another selective A2AR agonist 8 .In agreement with the in vitro data (new Fig. 3f, g), we found that PSB 0777-induced increases in plasma glucagon levels were abolished in α-GsKO mice, as compared to control littermates (new Supplementary Fig. 6c).These data clearly indicate that A2AR-stimulated glucagon secretion requires -cell Gs signaling.Appropriate changes were made in the revised manuscript (page 19, top).
10.The authors show that Gs signaling is crucial for GIP effects on glucagon secretion.Did the authors also investigate GLP-1?The mechanism underlying the inhibition of glucagon secretion by GLP-1 is controversial and the tools generated by the authors would provide valuable mechanistic insights.
The reviewer raises an interesting point.In fact, we are planning to carry out additional work focusing on the role of Gs in mediating the actions of GLP-1 on different islet cell types including -cells in a follow-up study.
11.The discussion about the source of adenosine in the islet is rather vague and could be improved.It seems as the authors favor an autocrine mechanism ("In theory…adenosine could be generated from beta cells"; "However, ... the enzyme that catalyzes …the conversion of ATP into adenosine is absent in mouse and human islets").Even if mouse and human islets lack the ectonucleotidase that converts ATP to adenosine, beta cells may be the source.
We fully agree with the reviewer.We therefore modified the part of the discussion that deals with this point as follows (page 25, center): "Despite lacking ecto-5' nucleosidase, mouse β-cells may also serve as a potential source of intraislet adenosine generated by the breakdown of intracellular ATP 9 .Minor: 12. Page 4, second para: The phrasing "direct glucose sensing is a key determinant of glucagon secretion" is somewhat unclear.Glucose "sensing" takes place both by neural mechanisms and by multiple cell types in the islets.Why not just "glucose is a key determinant"?
As requested by the reviewer, we now use the term "glucose is a key determinant" in the revised manuscript (page 4).13.P 4, third para: "dozen" should probably read "dozens".This has been corrected.14.P 8: "Incubation" does not seem to be a correct description."Exposure" or "perifusion" would be more appropriate for the protocol shown in Fig 1g .We replaced the term "incubation" with either "exposure" or "perifusion" at all relevant occasions.This has been corrected.16.On p 24, 1st paragraph: It is questionable if a study from 2010 should be called recent.
Regarding the 2010 paper: we removed the term "recent".

Reviewer #2
The authors of this manuscript explored a role for Gs in alpha-cells of pancreatic islets.They used a Gs-DREADD selectively expressed in these cells in a transgenic mouse model to probe the increasingly complicate physiology of the glucagon system.They also use an alpha-cell specific Gs knockout line of mice.The manuscript is well written in general and was a pleasure to read.
The characterization of the mouse models was appropriate and leads to confidence in asserting that they were alpha-cell specific.Controls for this were provided in the supplemental material.The experiments are well conducted and the conclusions well supported by the data provided.
Thank you for your very positive general comments.Comments 1) In the constitutive DREADD activity a potential confound?Might there be some homeostatic mechanisms that come into play because of this?
We agree with the reviewer that the constitutive activity of the Gs DREAADD (GsD) somewhat complicates the interpretation of the experimental data.To address this issue, we incorporated the following text into the Discussion section (page 24, top): "We noted that basal glucagon release was markedly increased in -GsD islets, as compared to control islets (Supplementary Fig. 1k).Previous studies have shown that the GsD designer receptor can signal, to a variable degree, in a ligand-independent fashion 10,11 .Thus, it is likely that the elevated glucagon levels observed with -GsD islets in the absence of an activating ligand are most likely caused by the constitutive signaling via -cell GsD.While this effect was easily detectable in -GsD islets in vitro, basal plasma glucagon levels were similar in -GsD mice and control littermates in vivo (Fig. 1a, d).This latter observation suggests that constitutive GsD signaling is unlikely to have a major impact on the outcome of the in vivo studies and that other physiological mechanisms, including, for example, circulating hormones and nutrients, paracrine factors, and neuronal pathways, limit constitutive GsD signaling in vivo." 2) In Figure 3a, why did SCH 442416 cause a decrease in glucagon levels on its own?Is there any indication that it is acting as an inverse agonist or a biased agonist for other pathways?This effect was also noted when measuring cAMP levels.Why was this effect not seen in Figure 3B?
The reviewer raises an interesting point.To address this issue, we incorporated the following text into the Discussion section (page 24, bottom, and page 25, top): "Somewhat surprisingly, treatment of WT mouse islets with a selective A2AR antagonist (SCH 442416) led to a pronounced reduction in glucagon secretion when glucose levels were low (Fig. 3a).As shown previously, intraislet adenosine 6 and -cell cAMP 7 levels are high in a low glucose environment.For this reason, the decrease in glucagon levels observed in the presence of SCH 442416 is most likely due to inhibition of glucagon release stimulated by adenosinemediated activation of -cell A2ARs.This mechanism also provides a likely explanation for the ability of SCH 442416 to greatly reduce cAMP levels at G3 (Fig. 3c).The glucagon release data shown in Fig. 3b were carried out at 12 mM glucose when intraislet adenosine levels are low 6 .As a result, SCH 442416 had no significant effect on basal glucagon secretion under these conditions.We are not aware of any published studies examining the ability of SCH 442416 to function as an inverse agonist or a biased agonist for other pathways.
3) Why weren't Figure 3c and 3d conducted in an identical way?
In Fig. 3c (G3), a subset of samples was incubated with SCH 442416 alone, without the UK 432097 agonist.In Fig. 3d (G12), this control was omitted since the experimental design included a SCH 442416 pre-incubation period (internal control).Omitting the "SCH 442416 alone curve" allowed us to study more islets at 12G, where cAMP measurements are noisier due to paracrine influences from -and -cell signaling (Merrins et al, unpublished data).
4) The immunofluorescence data in Figure 5c is not convincing.How about western blots?
To address this point, we modified Fig. 5c by enlarging specific areas to better visualize the presence (or absence) of Gs protein in -cells.Western blotting studies are problematic since they would require relatively large amounts of FACS-sorted -cells which only represent a small subpopulation of all islet cells.However, the qRT-PCR data shown in Fig. 5a convincingly demonstrate that mRNA coding for Gs is undetectable in -cells from α-GsKO mice.5d and 5e with the adenosine receptor agonist?

5) Why didn't the authors stimulate cells in Figure
We carried out these experiments.We incorporated the following text into the revised manuscript (page 19, top): "To further corroborate the involvement of α-cell Gs signaling in A2AR-stimulated glucagon secretion, we treated control islets and islets derived from α-GsKO mice (α-GsKO islets) with the A2AR agonist UK 432097 (50 nM) (Fig. 3f, g).We found that UK 432097-induced glucagon secretion was absent or nearly completely abolished in α-GsKO islets at both G3 and G12, respectively.In addition, we carried out vivo experiments with PSB 0777 (1 mg/kg, i.p.), another selective A2AR agonist 8 .In agreement with the in vitro data (Fig. 3f, g), PSB 0777-induced increases in plasma glucagon levels were abolished in α-GsKO mice, as compared to control littermates (Supplementary Fig. 6c).These data clearly indicate that A2AR-stimulated glucagon secretion requires -cell Gs signaling."6) In figure 6, why aren't there homeostatic alterations in insulin levels when glucagon levels are reduced?
The reviewer raises a very interesting point.To address this issue, we incorporated the following text into the Discussion section of the revised manuscript (page 27, top): "Although α-GsKO mice showed a significant decrease in plasma glucagon levels (Fig. 6a, d), this deficit did not cause any significant changes in plasma insulin levels (Fig. 6b, e).We speculate that chronic hypoglucagonemia causes compensatory changes involving other factors and neuronal pathways that can maintain normal plasma insulin levels.In agreement with this notion, previous studies demonstrated that the near-total ablation of -cells or suppression of -cell glucagon expression does have any discernible effect on plasma insulin levels in vivo [12][13][14] .In contrast, acute lowering of plasma glucagon levels due to activation of an inhibitory DREADD expressed in mouse -cells led to impaired insulin release in vivo 15 ."

Reviewer #3
The manuscript entitled "Novel mouse models establish a key metabolic role for α-cell Gs signaling" by Liu et.al demonstrate that Gs-coupled signaling impacts α-cell function.
Understanding alpha cell function and the regulation of glucagon secretion could have important implications for diabetes treatment so the topic is timely and relevant.The authors use chemogenetics to activate Gαs signaling within Gcg cells in lean and obese animals and study in vivo and ex vivo glucagon and insulin secretion.DREADD activation in both lean and obese animals stimulated glucagon and insulin secretion and improved IP glucose tolerance.The authors also employ several mouse models that disrupt Gs signaling (α-GsD and Gas) or Gs adenosine signaling (A2AR) and show that the A2A adenosine receptors play a role in stimulating glucagon secretion under low glucose conditions.Although there are interpretive issues, the authors state that the perifusion studies agree.Long-term disruption of Gαs signaling lowers Gcg expression, islet glucagon content, and consequently basal and glucagon responses to various stimuli.The authors conclude that Gαs is a novel target for regulating glucagon secretion.Although there is enthusiasm for the topic of research, there are weaknesses associated with the mouse model, the statistical analysis, and some issues with data interpretation.
Thank you for all your helpful comments which have led to a greatly improved revised manuscript.
Major comments: 1.The Gcg Cre utilized here has not been validated for brain expression.Gcg neurons are known to be expressed in the hindbrain and function to regulate various physiological functions including feeding patterns, body weight in some cases, motivation for feeding etc.More and more studies are being published on the impact of these neurons on physiology.Therefore, validation that the genetic manipulation has not targeted the brain is required.They easily could have done the HA tag staining should have been completed in the nucleus of the solitary tractwhy it was done in the hypothalamus instead is unclear.
The reviewer raises an important point.As requested, we studied whether -GsD mice also express the HA-tagged GsD designer receptor in the region of the nucleus of the solitary tract (NTS region).The use of an anti-HA antibody that we routinely use to visualize the expression of HA-tagged DREADDs in the brain (see, for example, Please see the new Supplementary Fig. 1c in the revised version of the manuscript.We also added the following short paragraph to the Results section (page 7, top): "Moreover, immunofluorescence staining of brain slices prepared from α-GsD mice failed to detect GsD expression in proglucagon-producing neurons of the nucleus tractus solitarius (NTS; Supplementary Fig. 1c)." 2. Were the data in Figure 1 analyzed with an ANOVA for repeated measures?If there is a group effect, but no time effect then statistically you can't say that DCZ actually increased glucagon or insulin.The statistics need to be redone with an ANOVA for repeated measures with an appropriate post hoc analysis for all graphs where time is a variable.
Yes, all data shown in Fig. 1a-f were analyzed via two-way repeated measure ANOVA for time and group with Bonferroni correction for pairwise post hoc comparisons of time.In Fig. 1d-f, we examined group difference at each time point separately because there was significant interaction between group and time.Moreover, we subjected all other data presented in this manuscript to a thorough statistical analysis following the guidance of the NIDDK Biostatistics Program.
3. Figure 1: Although it is not clear whether this is statistically significant, there is a 50% drop in glucagon with saline injection and with DCZ in the control animals.This drop is prevented in the GsD animals with DCZ-again it is not clear whether DCZ actually significantly increases glucagon at 15 min or whether it is simply preventing a drop in glucagon.This finding is very curious and needs further exploration/explanation.
As requested by the reviewer, the data shown in Fig. 1a-f were subjected to a thorough statistical analysis, following the advice of the NIDDK Biostatistics Program.Details regarding the statistical tests used are given in the figure legends.Based on the outcome of these analyses, we modified the text as follows ("Stimulation of α-cell Gs signaling leads to hyperglucagonemia and hyperinsulinemia in vivo") (page 7, center): "Similar to previous observations 15 , i.p. injection of control and α-GsD mice with saline or of control mice with DCZ resulted in reduced plasma glucagon levels 15 and 30 min after injection (p <0.0001; one-way repeated measures ANOVA, followed by post-hoc Bonferroni adjustment) (Fig. 1a, d).Although the precise mechanism underlying this phenomenon remains unclear, we speculate that the increase in blood glucose levels (see below; Fig. 1c, f) caused by the injection stress impairs glucagon secretion from -cells.Although there was a trend towards higher basal plasma glucagon level in -GsD mice (time 0; Fig. 1d), this effect failed to reach statistical significance (p= 0.085; Student's t-test at time 0).Importantly, DCZ treatment of -GsD mice led to a statistically significant increase in plasma glucagon levels (Fig. 1d), thus overcoming the inhibitory effect on glucagon release caused by the injection stress."4. Figure 1: Is there a statistical increase in glucose with the saline or DCZ injection?If so, then these studies are likely confounded by the animal's stress levels.
As requested by the reviewer, the data shown in Fig. 1a-f were subjected to a thorough statistical analysis, following the advice of the NIDDK Biostatistics Program.Details regarding the statistical tests used are given in the figure legends.Saline treatment of α-GsD mice and control littermates resulted in statistically significant increases in blood glucose levels (p <0.0001 at 15 and 30 min after injection (time 0); one-way repeated measures with time ANOVA for each group) (Fig. 1c).While this effect persisted in DZC-treated control mice, blood glucose levels remained unchanged after DCZ injection of α-GsD mice (Fig. 1f), most likely due the increase in plasma insulin levels observed with DZCtreated α-GsD mice (Fig. 1e) that "neutralized" the hyperglemic effect of the injection stress.
To address these points, we added additional text to the Results section (page 8, top). 5. Figure 1 and Supp Figure 1, perifusion studies: Is the relative increase in glucagon with the Ala or AAM greater than the control animals?It seems not.Thus, the increase in glucagon is simply due to an increase in basal concentrations.If this is the case then there seems to be an ex vivo and in vivo difference in glucagon responses that needs to be discussed.Further, it is unclear why a doubling of glucagon under "basal conditions" is visible in perifused islets but not in vivo.The fact that the insulin is normal at baseline and is higher with amino acid stimulus even though the relative increase in glucagon is similar dissociates glucagon and insulin responses to amino acids under these conditions.This becomes hard to see with the expanded scale in Figure 1g.The components of the perifusion experiments should be separated out so the data can be visualized on an appropriate scale for Fig 1g .In fact, is it the scale or did the control alpha cells not increase glucagon secretion in response to alanine?
As requested by the reviewer, we subjected the perifusion data shown in Fig. 1 and Suppl.Fig. 1 to a thorough statistical analysis, following the advice of the NIDDK Biostatistics Group.Details regarding the statistical tests used are given in the figure legends.
Question: "Is the relative increase in glucagon with the Ala or AAM greater than the control animals?"To correct for differences in basal hormone secretion, we calculated area of the curve (AOC) values by subtracting the areas under or over the baseline 3 .This type of analysis is the method of choice when baseline levels between two or more experimental groups differ 3 .In the presence of alanine, DCZ treatment of -GsD islets caused a statistically significant increase in glucagon release at both G12 and G3, as compared to control islets (Fig. 1g, h in the revised manuscript).We also subjected the insulin secretion data shown in Fig. 1h of the original manuscript to the same type of analysis.We found that insulin responses were significantly elevated at G12 (but not at G3) in -GsD islets, as compared to control islets (new Fig. 1i, j).In the absence of DCZ, amino acid-induced glucagon and insulin release were similar in -GsD and control islets (revised version of Suppl.Fig. 1k, l).We made appropriate changes in the revised manuscript.Question: "Further, it is unclear why a doubling of glucagon under "basal conditions" is visible in perifused islets but not in vivo."As stated by the reviewer, basal glucagon release at both G3 and G12 was significantly higher in -GsD islets, as compared to control islets (Fig. 1g and Supplementary Fig. 1k).This observation is consistent with previous findings that the GsD designer receptor shows a certain degree of constitutive activity under distinct experimental conditions 11,16 .As pointed out in the discussion section of the revised manuscript, previous studies have shown that the GsD designer receptor shows a certain degree of constitutive activity under distinct experimental conditions 11,16 .Most likely, this increase in ligand-independent signaling is responsible for the observation that basal glucagon secretion is elevated in -GsD islets.In contrast, in vivo studies showed that basal plasma glucagon levels were not significantly different between -GsD mice and control littermates (Fig. 1a, d).It is likely that chronic activation of -cell Gs caused by GsDmediated constitutive signaling triggers compensatory changes in vivo that can maintain normal plasma glucagon levels and euglycemia.In agreement with this notion, circulating glucagon levels are regulated by various neuronal pathways and other factors (e.g.hormones and other circulating bioactive agents) that are not present in isolated islets 17,18 .
We address this issue in the Discussion section of the revised manuscript (page 24, top).We also made changes in the Results section (page 8, bottom).
Question: "In fact, is it the scale or did the control alpha cells not increase glucagon secretion in response to alanine?" Thank you raising this point.The scale used in the original version of Fig. 1g did not reveal the small increase in glucagon secretion triggered by treatment of control islets with alanine.To better visualize this response, we added an insert focusing on alanine-stimulated glucagon secretion in the revised version of Fig. 1g (for a statistical analysis of the data, please see Fig. 1h).

Statistical analysis of the perifusion studies needs to be reported.
As requested by the reviewer, we subjected all islet perifusion data to a thorough statistical analysis.Statistical tests were chosen based on the advice of the NIDDK Biostatistics Group.Details regarding the statistical tests used are given in the figure legends.To quantitate changes in hormone levels, we calculated AOC values that correct for altered hormone baseline levels 3 .This type of analysis is the method of choice when baseline levels between two or more experimental groups differ 3 .
7. Body weight of Adora2af/f Gcg cre mice is needed.
We added body weight data to Suppl.Fig. 4 (new panel 'g').Body weights were similar in control and mutant mice.
8. Interpretation of the 2dg data in the alpha-A2AR KO mice is not accurate.These mice had decreased glucagon and increased glucose responses to 2DG.Under glucoprivic conditions, increases in glucagon function to stimulate hepatic glucose production and increase plasma glucose.Since insulin levels are the same, then this clearly indicates that the insulin to glucagon ratio should favor a suppression of hepatic glucose production.Therefore, some other counterregulatory factor is regulating the glucose response to 2DG in the face of inadequate glucagon levels in these animals.This suggests off-target impact on sympathetic neurons.Plasma catecholamines, which also potently respond to 2DG and regulate glucose homeostasis should be assessed.
Like the reviewer, we were also puzzled by these findings.For this reason, we decided to repeat this experiment with a new set of mice that we specifically generated to revisit this issue.Using this new batch of mice, we were unable to replicate the data presented in original version of the manuscript (Fig. 4g, h; probably an artifact).The new data show that 2-DG-induced increases in plasma glucagon, plasma insulin, and blood glucose levels were not significantly affected by the lack of -cell A2ARs.We therefore replaced Fig. 4g, h (original manuscript) with a suppl.figure (new Suppl.Fig. 4q-s).displaying these new data We also changed the text in the Results section accordingly in the revised manuscript (page 16, center).9.There are more 2DG interpretive issues in Figure 7. Specifically in the second part of this sentence: "The glucopenia-induced increases in blood glucose levels were significantly more pronounced in a-GsKO mice (Fig. 7d), probably due to decreased plasma insulin levels (Supplementary Fig. 7a, b)."The 2dg-induced (and it's not 2DG it's the increase in glucose that drives the increase in insulin) insulin secretion was not reduced, it simply returned to baseline quicker.Thus, the conclusion that α-cell Gs signaling contributes to the counter-regulatory glucagon responses is not accurate.This is another specific example where a repeated measures anova for time is necessary so that the responses over time can be accurately assessed and interpreted.
Thank you for these helpful comments.A two-way ANOVA for repeated measures with Bonferroni post hoc test revealed a significant group effect, although the time-by-group interaction was not statistically significant.Based on this analysis and the reviewer's comments, we rephrased the manuscript text as follows (page 21, center): "In α-GsKO mice, the 2-DG-dependent increase in glucose-driven insulin secretion returned to baseline more rapidly than in control littermates (Supplementary Fig. 8a, b).It is likely that this effect is responsible for the elevated blood glucose levels observed with 2-DG-treated α-GsKO mice, as compared to 2-DG-treated control littermates (Fig. 7d)." 10.The interpretation of the glucagon responses in the alpha-GsKO mice is also curious.Gcg expression is decreased, islet glucagon content is decreased.So if there is limited gene and protein, then it is not surprising that there is no glucagon response to glucagon secretagogues.It also brings to question why the KO of alpha cell Gs leads to a decrease in Gcg expression in the first place.
Regarding the first part of the reviewer's comments: As stated by the reviewer, Gcg RNA levels and glucagon content are markedly decreased (by ~70-80%) in -GsKO islets, as compared to control islets.It is highly likely that these deficits make a major contribution to the functional impairments displayed by the -GsKO islets.However, UK 432097-and isoproterenol-stimulated glucagon secretion was absent or nearly abolished in α-GsKO islets (revised Fig. 3f, g and Fig. 5d, e, respectively).These data indicate that the lack of -cell Gs signaling also contributes to the functional deficits caused by -cell Gs deficiency.We now briefly discuss this issue in the revised version of the manuscript (page 27, bottom).
Regarding the second part of the reviewer's comments: To investigate the mechanism underlying the decrease in Gcg expression caused by -cell Gs deficiency, we carried out additional studies with cultured mouse -cells (-TC6 cells) and isolated mouse islets.Treatment of -TC6 cells with PKI 14-22 (10 μM), a highly selective inhibitor of PKA, a protein kinase activated by Gs signaling, led to significantly reduced Gcg RNA levels (new Supplementary Fig. 7g).We now address this point in the revised version of the manuscript (page 20, center; page 27, bottom ,and page 28, top).We also incorporated the following paragraph into the Discussion section: "The activity of the rodent Gcg promoter is under the control of several regulatory factors, including a cAMP response element (CRE) (reviewed in 19,20 .The promoter of the rodent Gcg gene contains a CRE sequence that is activated by cAMP-dependent protein kinase A, resulting in enhanced Gcg transcription 21,22 .This observation is consistent with our finding that impaired -cell Gs/cAMP signaling leads to reduced Gcg RNA levels (Fig. 5k) and decreased pancreatic glucagon content in a-GsKO islets (Fig. 5g, h).The latter finding also provides an explanation for the observation that treatment of -GsKO islets with KCl or with an agonist that acts on Gqcoupled V1bRs resulted in impaired increases in glucagon secretion (Fig. 5f, 7h).In agreement with these published data 21,22 , we showed that treatment of cultured mouse -cells (TC6 cells) with PKI 14-22, a highly selective inhibitor of PKA, resulted in significantly decreased Gcg RNA levels (Supplementary Fig. 7g).In sum, these findings highlight the importance of basal -cell Gs signaling in maintaining proper Gcg transcription and glucagon content in pancreatic islets." 6. Disruption of α-cell Gs signaling suppresses the expression of the Gcg gene, leading to reduced glucagon synthesis and storage.This deficit is most likely responsible for the hypoglucagonemia phenotype displayed by the α-GsKO mice.These findings highlight the importance of basal -cell Gs signaling in maintaining proper Gcg transcription and glucagon content in pancreatic islets.
7. We also present data strongly suggesting that -cell Gs signaling is required for the proper release of glucagon and insulin after consumption of a mixed meal.
8. We demonstrate that -cell Gs signaling plays an important role in mediating the counterregulatory stimulation of glucagon secretion during hypoglycemic and glucopenic states.
The revised manuscript is much improved and the authors have answered satisfyingly to most of the referee comments.However, this reviewer is concerned that the authors were unable to replicate some of the findings reported in the original version of the manuscript, when experiments were repeated as a consequence of comments from the referees (point 8 by both referee 1 and 3).No explanation for the discrepancy is provided other than "probably an artifact".This is unsettling and the question arises whether also other experiments may be affected by similar artifacts.

OUR RESPONSE
We are glad that the reviewer considers our manuscript much improved (after a very positive initial review), stating that "the authors have answered satisfyingly to most of the referee comments." The specific experiment that the reviewer is referring to deals with the following observation, as described by rev. 1 in her/his original review (point 8): "P 15/Fig 4g,h: 2-DG administration triggers glucagon secretion, which is impaired in the a-A2AR-KO.Yet, the resulting hyperglycemia is more pronounced in the KO, despite similar insulin levels.This seemingly confusing observation deserves to be discussed."Like the reviewer, were also puzzled by these data since they were not consistent with all other experimental data contained in the manuscript.For this reason, we decided to repeat this particular experiment.The repeat experiment showed that the effect of 2-DG treatment on hyperglycemia was similar in -A2AR-KO and control mice (Suppl.Fig. 4q-s in the revised manuscript).Please note that this experimental outcome is not central to the key conclusions outlined above.
We would also like to note that that the outcome of in vivo studies in mice is affected by numerous external parameters (e.g.mouse housing conditions, litter size, precise time of the day when a specific experiment is done, temperature and humidity in the animal facility, prior presence of technical staff in the animal room, etc.) which occasionally leads to unexpected results.My lab has more than 25 years of experience in analyzing metabolic parameters in mutant mice in vivo.Our manuscript describes a very large number of in vivo experiments.For this reason, the likelihood that a specific experiment (which, in this case, is not critical for the As suggested by Reviewer 1, we reworded the text dealing with this matter as follows: "Although the precise mechanism underlying this phenomenon remains unclear, it is possible that the stress of the i.p. injection is confounding basal hormonal levels."Page 7, bottom paragraph.7d: There are still no statistical comparisons for given time points reflected on the graph.If the 2way ANOVA reveals a significant time x genotype interaction then the post hoc analysis will reveal at what time points this is significant.The time points where glucose higher in the alpha-GsKO mice needs to be denoted on the graph panel with an asterix over the time point.

OUR RESPONSE
As requested by the reviewer, we now added a p value (0.0289) to the 120 min time point (no other time points showed statistically significant differences; Fig. 7d).The Nature Commun instructions for authors recommend showing actual p values in the figures, rather than asterisks.
3. In the results and in reference to the insulin levels after 2DG, please correct the line prior to the highlighted text which still incorrectly says "2-DG-induced insulin secretion….." ."At the same time, 2-DG-induced insulin secretion was also significantly reduced (Supplementary Fig. 8a, b).In α-GsKO mice, the 2-DG-dependent increase in glucose-driven insulin secretion returned to baseline more rapidly than in control littermates (Supplementary Fig. 8a, b).It is likely that this effect is responsible for the elevated blood glucose levels observed with 2-DG-treated α-GsKO mice, as compared to 2-DG-treated control littermates (Fig. 7d)."

OUR RESPONSE
We apologize for this oversight.As requested by the reviewer, we rephrased this sentence in the re-revised version of the manuscript (page 21, center paragraph).
4. There remains a huge concern over the conclusion that isoproterenol-stimulated glucagon secretion requires alpha-cell Gs signaling.The alpha-GsKO animals have limited Gcg gene or protein.How would UK 432097 stimulate glucagon if there is none there to stimulate?Putting it another way, what would the conclusion be if UK 432097 stimulated glucagon secretion?Where would the authors think that glucagon be coming from in that case?

OUR RESPONSE
The reviewer raises an important point that we should have addressed in more detail in the revised version of the manuscript.The following observations/comments should be able to allay the reviewer's concerns: The -adrenergic, A2A adenosine, and GIP receptors are Gs-coupled receptors (PMID: 38123151).We mention this fact for the -adrenergic and A2A adenosine receptors in the main text of the manuscript (page 5, first paragraph; page 11, 1st paragraph; page 17, line 9; page 24, 2nd paragraph).Moreover, for increased clarity, we added the following sentence on page 21 (4th and 5th line from the bottom): "Previous work has shown that the GIP receptor is selectively linked to Gs (ref.60)".Thus, as expected, studies with isolated islets showed that the lack of -cell Gs virtually abolished the ability of GPCR agonists acting on -cell -adrenergic, A2A adenosine, and GIP receptors (isoproterenol, UK 432097, and GIP, respectively) to promote glucagon release (Figure 5d, e; 4b, 7h).In contrast, treatment of α-GsKO islets with a V1bR agonist (d[Leu 4 ,Lys 8 ]VP) still resulted in a significant stimulation of glucagon secretion (Figure 5f; G12).In contrast to the adrenergic, A2A, and GIP receptors, the V1bR is a Gq/11-coupled receptor that releases glucagon by activating -cell Gq/11 signaling (PMID: 34752420).The ability of the V1bR to promote significant glucagon secretion in the absence of -cell Gs clearly indicates that there is still a substantial amount of releasable glucagon left in α-GsKO mice (Figure 5h).Moreover, KCl retained the ability to stimulate glucagon section in α-GsKO islets, although with reduced efficacy (see, for example, Figure 5f, 7h).In agreement with the in vitro data, α-GsKO mice retained the ability to stimulate the release of glucagon in vivo under glucopenic conditions, although this response was reduced relative to control littermates (Fig. 7c).Finally, pancreata from α-Gs KO mice still contain a considerable amount of glucagon (Figure 5h) which can serve as the source of glucagon in response to non-Gs-dependent stimuli.
These observations clearly indicate that the relative inability of GPCR agonists acting on cell -adrenergic, A2A, and GIP receptors to promote glucagon release from α-Gs KO islets is due to the selective inactivation of -cell Gs signaling, in combination with the reduced amount of glucagon stored by α-Gs KO islets.
We added an extra paragraph to the Discussion section of the re-revised version of the manuscript to clarify this matter (page 26, bottom and page 27, top).

11 .
It would be interested to know how somatostatin regulation of the alpha cell fits into this story.Minor Comments: 12. Supp Fig 1, Panel B should show individual channels of all fluorophores used. 1 01-25-2024 Response to the Referees' comments Nature Communications NCOMMS-23-26486, revised version TITLE: " Novel mouse models establish a key metabolic role for α-cell Gs signaling" by Liu Liu et al.
; Fig.. 4a)." 2. The immunofluorescence images in Suppl Fig 1b is too dim, at least in the pdf version of the manuscript.That quality is insufficient and must be improved.
Fig 1b in ref.PMID: 26743492) failed to yield a detectable immunofluorescence signal in the NTS region, indicative of the lack of GsD expression in the NTS or of very low GsD expression levels below the detection threshold of the protocol used.